Cell Reports

Resource
Interactome of Two Diverse RNA
Granules Links mRNA Localization
to Translational Repression in Neurons
Renate Fritzsche,
1,5
Daniela Karra,
1,2,5,6
Keiryn L. Bennett,
3
Foong yee Ang,
1,4
Jacki E. Heraud-Farlow,
1,7
Marco Tolino,
1
Michael Doyle,
1
Karl E. Bauer,
1,4
Sabine Thomas,
1,2,4
Melanie Planyavsky,
3
Eric Arn,
1,2
Anetta Bakosova,
1
Kerstin Jungwirth,
1,2
Alexandra Ho rmann,
1,8
Zsoa Pal,
1
Julia Sandholzer,
1,11
Martina Schwarz,
1,9
Paolo Macchi,
1,2,10
Jacques Colinge,
3
Giulio Superti-Furga,
3
and Michael A. Kiebler
1,2,4,
*
1
Department of Neuronal Cell Biology, Center for Brain Research, Medical University of Vienna, 1090 Vienna, Austria
2
Max-Planck-Institute for Developmental Biology, 72076 Tu bingen, Germany
3
CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, 1090 Vienna, Austria
4
Department for Anatomy & Cell Biology, Ludwig-Maximilians-University, 80336 Munich, Germany
5
These authors contributed equally to this work
6
Present address: Paul-Ehrlich-Institute, 63225 Langen, Germany
7
Present address: Max F. Perutz Laboratories, University of Vienna, 1030 Vienna, Austria
8
Present address: Boehringer Ingelheim, 1121 Vienna, Austria
9
Present address: GMI, 1030 Vienna, Austria
10
Present address: Centre for Integrative Biology (CIBIO), University of Trento, 38060 Mattarello (TN), Italy
11
Deceased
*Correspondence: michael.kiebler@med.uni-muenchen.de
http://dx.doi.org/10.1016/j.celrep.2013.11.023
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works
License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are
credited.
SUMMARY
Transport of RNAs to dendrites occurs in neuronal
RNA granules, which allows local synthesis of spe-
cic proteins at active synapses on demand, thereby
contributing to learning and memory. To gain insight
into the machinery controlling dendritic mRNA local-
ization and translation, we established a stringent
protocol to biochemically purify RNA granules from
rat brain. Here, we identied a specic set of inter-
actors for two RNA-binding proteins that are known
components of neuronal RNA granules, Barentsz
and Staufen2. First, neuronal RNA granules are
much more heterogeneous than previously antici-
pated, sharing only a third of the identied proteins.
Second, dendritically localized mRNAs, e.g., Arc
and CaMKIIa, associate selectively with distinct
RNA granules. Third, our work identies a series of
factors with known roles in RNA localization, transla-
tional control, and RNA quality control that are likely
to keep localized transcripts in a translationally
repressed state, often in distinct types of RNPs.
INTRODUCTION
Following transcription, RNA is spliced, modied, and exported
into the cytoplasmin ribonucleoprotein particles (RNPs). mRNAs
are then either immediately translated by ribosomes or transla-
tionally repressed and transported to their nal site of function,
where they can be locally translated upon demand (Doyle and
Kiebler, 2011; Holt and Bullock, 2009; Martin and Ephrussi,
2009; St Johnston, 2005). In the CNS, a subset of mRNAs is
transported to dendrites, where they are translated at the
synapse. It is widely believed that such local synthesis of new
proteins contributes to synaptic plasticity, learning, and memory
(Costa-Mattioli et al., 2009; Sutton and Schuman, 2006). How-
ever, little is known about the underlying mechanisms.
Several studies have now reported the purication of various
RNA granules from different sources (Brendel et al., 2004; Elvira
et al., 2006; Jnson et al., 2007; Maher-Laporte et al., 2010;
Villace et al., 2004). To date, these studies yielded a large list
of protein interactors with very little overlap between associated
proteins. Due to the large variance, our understanding of the
molecular composition of neuronal RNA granules (Anderson
and Kedersha, 2006; Kiebler and Bassell, 2006) including their
functions remains incomplete.
In order to further our knowledge into the molecular mecha-
nism of mRNA localization in the CNS and to investigate a
possible link with translational control and mRNA decay, we
puried two types of neuronal RNA granules from rat brain using
two established markers for neuronal RNA granules: Staufen2
(Stau2) and Barentsz (Btz or CASC3, MLN51). Work fromvarious
organisms demonstrated that the best molecular markers to
follow RNP transport are Staufen proteins (Ko hrmann et al.,
1999; Martin et al., 2003; Tang et al., 2001; Zimyanin et al.,
2008) and Barentsz (Macchi et al., 2003; Palacios et al., 2004;
van Eeden et al., 2001). Using this multistep biochemical
approach, we report the protein interactome of both Btz- and
Cell Reports 5, 17491762, December 26, 2013 2013 The Authors 1749
Stau2-RNPs from rat brain: 84 and 65 proteins associate with
Btz and Stau2, respectively, with only a third of the proteins
being shared between both RNP complexes, e.g., the RNA
helicase Upf1, FMRP, and Pur-a among others. Furthermore,
the dendritic mRNAs Arc and CaMKIIa associate preferentially
with Btz- and Stau2-RNPs, respectively. These results argue
for neuronal RNA granule heterogeneity. Furthermore, Btz- and
Stau2-RNPs are distinct from processing bodies or stress
granules, representing other types of neuronal RNA granules.
In addition, components of the exon junction complex (EJC),
eIF4AIII, Y14, and Magoh are only found in Btz- but not in
Stau2-RNPs. In contrast, the cap-binding protein CBP80 is
found in both Btz- and Stau2-RNPs and colocalizes with Stau2
in neuronal dendrites. Together with the fact that several transla-
tional repressors associate with both RNPs, our results suggest
that these RNPs are translationally stalled. This experimentally
conrms a widely accepted hypothesis that transcripts might
be translationally repressed during transport preventing their
expression elsewhere (St Johnston, 2005).
Taken together, the Btz and Stau2 interactome suggest that
factors involved in RNA localization, translational control, and
RNA quality control participate in keeping localized transcripts
in a translationally repressed state. This study therefore comple-
ments previous work on the RNA level showing that localized
transcripts are often found in distinct types of RNPs (Amrute-
Nayak and Bullock, 2012; Batish et al., 2012; Mikl et al., 2011;
Tu bing et al., 2010). It is tempting to speculate that neuronal
RNA granules are much more heterogeneous and possibly
more dynamic than previously anticipated.
RESULTS
Isolation of Endogenous RNPs
Our goal was to isolate endogenous RNPs fromrat brain extracts
to decipher the molecular composition of neuronal RNAgranules
(Kiebler and Bassell, 2006) in order to gain more insight into
the underlying mechanism of dendritic mRNA localization.
Therefore, we established a multistep biochemical isolation
protocol for neuronal RNA granules or RNPs (Figure 1A) based
on our previous work (Mallardo et al., 2003). This improved
approach comprises three distinct steps: (1) the generation of
soluble brain lysates; (2) the enrichment of RNA granules
via density gradient centrifugation; and (3) preparative scale
immunoprecipitations (IPs) using mono-specic, afnity-puried
antibodies that allow the afnity purication of endogenous
RNA-protein complexes. In detail, soluble S20 supernatants
were generated from E17 rat brain lysates that are depleted for
intact cells, cell debris, and large membrane-bound organelles
(Mallardo et al., 2003), often yielding high background in IP
experiments due to their binding to solid matrices, e.g., protein
A Sepharose (data not shown). Next, Optiprep density gradient
sedimentation (Kiebler et al., 1999b) was used to enrich for intact
RNA-containing Stau2- and Btz-RNPs, while separating them
from the main protein peak (Figure 1B) as well as from the
endoplasmic reticulum (as shown by the absence of the ER
marker calnexin) (Figure 1C). The cofractionation of four known
markers for neuronal RNPs, e.g., Btz (Macchi et al., 2003),
Stau2 (Goetze et al., 2006; Tang et al., 2001), Stau1 (Kanai
et al., 2004; Mallardo et al., 2003), and Poly(A)-binding protein
1 (PABP1) in fractions 47 of the Optiprep gradient identied
dense RNPs. The presence of PABP1 furthermore suggests
that these RNPs are still intact containing 3
0
-polyadenylated
mRNA. Pretreatment of S20 supernatants with RNase shifted a
signicant portion of the Stau2- and Btz-RNPs toward lighter
Optiprep fractions on top of the gradient conrming them as
bona de RNPs (Figure 1D) providing further evidence that the
isolated particles are intact and contain RNAs.
We then employed preparative scale IPs using mono-specic,
afnity-puried antibodies that recognize two components of
neuronal RNA granules: Btz and Stau2 (Kiebler and Bassell,
2006). We have previously generated specic polyclonal anti-
bodies directed against Stau2 (Goetze et al., 2006; Zeitelhofer
et al., 2008) and Btz (Macchi et al., 2003; Zeitelhofer et al.,
2008) (Figure S1; data not shown) that not only recognize their
native antigen(s), but also precipitate endogenous RNPs (Fig-
ure 2; data not shown). These antibodies thus allow us to specif-
ically enrich for neuronal RNA granules by afnity purication.
Antibodies were chemically crosslinked to the matrix (Sisson
and Castor, 1990). Btz and Stau2 preimmune sera (PIS) from
the same rabbits served as control. Nonspecic binding to the
matrix was blocked by pretreating beads with tRNA and BSA
(Harlow and Lane, 1988). Elution of the RNPs from the matrix
was performed in a sequential manner: (1) RNA-dependent
interactors were eluted by treatment with RNases A and T1
(RNase Eluate); and (2) protein-dependent interactors were
subsequently eluted by low pH (Glycine Eluate). Both eluates
were separated by SDS-PAGE and proteins were silver stained
(Figures 2A and 2B). Compared to the respective control lanes
(PIS-IP for Btz, A, or for Stau2, B), a number of proteins sig-
nicantly enriched in both IPs indicating that we were able to
isolate high molecular weight RNPs.
Systematic Identication of Protein Interactors
Entire gel lanes were excised, 20 regions digested in situ with
trypsin, and the samples analyzed by liquid chromatography
mass spectrometry (LC-MS). Proteins were identied by search-
ing the MS-generated data against both the mouse and rat
International Protein Index protein sequence databases. The rat
database was also searched to identify (by homology) unse-
quenced mouse proteins that were not included in the more
complete mouse database. Only proteins that were present in
all three independent experiments and identied by at least
two peptides (or by three peptides in n-1 experiments) were
considered as true positives (Table S1, Positives). Importantly,
proteins that were also identied in the respective control IPs
were excluded from further analysis (Table S2, Negatives).
In total, 84 proteins were identied in Btz IPs compared to
65 proteins in Stau2 IPs (Figure 2C; Table S1, Positives). Only
29 proteins (35% for Btz versus 45% for Stau2) are shared
between the two types of RNPs, providing further evidence
that both proteins are not simply part of the same RNP and that
neuronal RNA granules are much more heterogeneous. This
conrms immunouorescence data showing that Btz and Stau2
do not show signicant colocalization in dendritic RNPs in
mature neurons (Figure S2), thus implying that RNPs are possibly
more dynamic and heterogeneous than previously anticipated.
1750 Cell Reports 5, 17491762, December 26, 2013 2013 The Authors
To gain insight into the various functions of the identied
proteins, we performed gene ontology (GO) term analysis using
DAVID (http://david.abcc.ncifcrf.gov). This revealed that ve out
of the six top categories were related to RNA binding (Figure 2D)
as many of the identied proteins are known RBPs: 34/84 for
Btz and 31/65 for Stau2: e.g., FMRP, Pur-a, Stau1 (see Table
S3 for full list). Among these is a set of RNA helicases
that specically enrich with either Btz- or Stau2-RNPs: Ddx1,
eIF4AIII/Ddx48, Mov10. Most signicantly, however, many of
the identied RBPs have been implicated in RNA localization
including translational control or RNP biogenesis: e.g., FMRP,
Pur-a, Stau1, and eIF4AIII (Table S3).
Identication of Cargo mRNAs for Btz- and Stau2-RNPs
To further corroborate our ndings that neuronal RNA granules
are linked to mRNA localization, we investigated whether six
established dendritically localized mRNAs are associated with
either of the RNPs. Total RNA was isolated from the IP, reverse
optiprep density gradient 15 - 30%
y v a e h t h g i l
main protein peak
C
o
o
m
a
s
s
i
e
main protein peak
Btz
Stau2
62
59
52
Stau1
PABP1
CNX
L7a
S20 1 2 3 5 4 6 7 8 10 9 11
optiprep density gradient 15 - 30%
light heavy - RNase
W
e
s
t
e
r
n

b
l
o
t
W
e
s
t
e
r
n

b
l
o
t
S20 1 2 3 5 4 6 7 8 10 9 11
+ RNase
D
Btz
Stau2
62
59
52
Stau1
PABP
CNX
L7a
B
C
optiprep density gradient 15 - 30%
light heavy
A
Figure 1. Btz and Stau2 Granules Are RNA-Containing RNPs
(A) Schematic outline of the Btz-RNP purication. The isolation of endogenous Stau2-containing RNPs was essentially the same, except that Stau2 antibodies
were used (see also Figure S1).
(BD) Size fractionation of neuronal RNA granules using Optiprep density gradient centrifugation. 1.5% of each fraction was separated by SDS-PAGE and either
stained with Coomassie blue (B) or blotted onto nitrocellulose and probed with the antibodies indicated on the left (C and D). Btz- and Stau2-containing RNPs
are enriched in fractions 46 (Btz) or fractions 57 (Stau2), respectively. Both RNP peaks are clearly separated from free soluble proteins (labeled main protein
peak in (B) on top of the gradient. The presence of Stau1 as well as the poly(A)-binding protein 1 (PABP1) indicates that the isolated RNPs were still intact.
Calnexin (CNX) and ribosomal protein L7a served as markers to follow the fate of ER and ribosomes, respectively. (D) Btz- and Stau2-containing complexes
represent true RNA granules. Soluble E17 rat brain lysates were pretreated with RNase A + T1 before loading onto the gradients causing disassembly of RNPs.
This shifts all RNP markers, e.g., Btz, Stau2, Stau1, and PABP1, toward lighter fractions on top of the gradient.
See also Figure S1.
Cell Reports 5, 17491762, December 26, 2013 2013 The Authors 1751
D
P-value Original Corrected P-value Original Corrected Shared
1
2
3
4
RNP complex 2.1E-17 22 22 2.3E-21 23 23 12
5 RNP biogenesis 2.6E-03 5 7 5.9E-02 3 5 1
6 (RNA) Helicase activity 2.3E-03 5 5 8.9E-03 4 4 0
7 Translation initiation factor activity 2.4E-03 3 2 1.8E-02 2 1 1
8 Ribosome 9.1E-04 6 6 2.6E-05 7 7 6
9 Gene silencing by miRNA 0 1 4.0E-02 2 2 0
2 n e f u a t S z t B
n.a.
RNA binding 8.0E-19 27 34 6.3E-16 22 31 14
Nucleic acid binding 2.8E-11 38 38 2.5E-09 30 30 14
RNA localization 1.8E-04 5 12 4.0E-05 5 9 6
RNase Eluate Glycine Eluate RNase Eluate Glycine Eluate
Stau2-IP PIS-IP Stau2-IP PIS-IP Btz-IP PIS-IP Btz-IP PIS-IP
B A
C
55 36 29
Stau2 Btz
84 65 proteins vs Total:
Figure 2. Molecular Composition of Btz- and Stau2-Containing RNPs
(A and B) Preparative IPs from pooled Optiprep gradient fractions (F4-6 for Btz and F5-7 for Stau2, respectively) were performed using Btz (A) or Stau2 (B)
antibodies (see also Figures S1 and S2). Control IPs using corresponding preimmune sera (PIS) were performed in parallel. Immunoprecipitates were eluted in two
steps (see Figure 1A): rst with an RNase A/T1-containing buffer (RNase Eluate) followed by a low pH step (glycine pH 2.5; Glycine Eluate). The resulting eluates
were separated by SDS-PAGEand visualized by silver staining. Representative examples are shown. Individual lanes were excised completely and processed for
LC-MS (see Table S1 for a complete list of identied interactors and Table S2 for a complete list of proteins that were found in the corresponding PIS-IP lanes).
Please note the enrichment of associated proteins in the Btz- (labeled with arrowheads) and Stau2-IP lanes compared to the corresponding PIS-IP lanes.
Molecular weight markers are shown on the left. Asterisks mark immunoglobulin G chains that are coeluted by glycine.
(C) Graph indicating the number of proteins identied in Btz- and/or in Stau2-RNPs.
(D) Gene ontology (GO) term analysis of the isolated proteins. Proteins were identied by automated database searching as described in Experimental
Procedures against both murine and rat databases, ofcial gene symbols were used to perform GO term analysis using DAVID (http://david.abcc.ncifcrf.gov).
For each GO term category, the count (number of proteins identied) and the respective p value are indicated. Proteins with known functions that have
been overlooked by GO term were added or false annotations deleted. On the right, the number of shared components between Btz- and Stau2-RNPs are listed
(see Table S3 for a complete list).
Original, components identied by DAVID; corrected, missing proteins were added or deleted if falsely annotated; n.a., not applicable. See also Figure S1,
Tables S1, S2, and S4.
1752 Cell Reports 5, 17491762, December 26, 2013 2013 The Authors
transcribed, and subjected to quantitative RT-PCR (qRT-PCR).
The amplied mRNAs were then quantied relative to the input
and control IPs for both Btz and Stau2 (Figure 3). The activity-
regulated cytoskeletal protein 1 (Arc) mRNA and the Lim-
domain-containing kinase 1 (Limk1) mRNA were specically
enriched in the Btz IP (17.4- and 1.6-fold enrichment, respec-
tively). In contrast, Ca
2+
/Calmodulin-dependent kinase II alpha
(CaMKIIa) mRNA was specic to Stau2 (2.9-fold), whereas
Lysophospholipase 1 (Lypla1) mRNA was found in both RNPs
(3.0- and 2.0-fold enrichment in Stau2 and Btz IPs, respectively).
These differences in the RNA composition conrmthe heteroge-
neity of the two RNPs previously observed on the protein level.
Furthermore, the presence of Arc mRNA in Btz-RNPs and
CaMKIIa mRNA in Stau2-RNPs provides experimental evidence
that we have succeeded in enriching for neuronal RNA granules
that are involved in RNA localization. Interestingly, eIF4AIII,
another component of the EJC (Le Hir and Se raphin, 2008),
has been previously linked to dendritic localization and stability
of Arc mRNA in neurons, providing further validity for its
association with Btz-RNPs (Giorgi et al., 2007). In addition to
this candidate approach, we recently identied Btz- and
Stau2-associated RNAs by microarray and found that only a
small set of mRNAs are shared between Btz- and Stau2-RNPs
(Heraud-Farlow et al., 2013; data not shown).
The EJC Is Part of Btz-, but Not Stau2-RNPs
From the LC-MS data, we noted that eIF4AIII and Magoh were
positive interactors for Btz; however, neither were found with
Stau2 (Table S1). For Btz, this was expected as it is a core
component of the EJC (Andersen et al., 2006; Bono et al.,
2006; Le Hir and Se raphin, 2008) and has been previously linked
to mRNA localization (Macchi et al., 2003; van Eeden et al.,
2001). The fourth EJC core component, Y14, was detected in
Btz-RNPs but was below the threshold to make the cutoff. The
EJC assembles on newly formed mRNAs concomitant with
splicing and remains associated with that message until it is
translated in the cytoplasm. The EJC is important for several
stages of mRNA fate, including nuclear export, mRNA stability,
localization, and translation (Le Hir and Se raphin, 2008). Further-
more, eIF4AIII has been previously implicated in dendritic
localization of Arc mRNA in neurons (Giorgi et al., 2007). There-
fore, we decided to independently validate whether EJC
members were part of our neuronal Btz- and Stau2-RNPs. By
western blot, all three core proteins of the EJC were found to
be present in Btz- but not in Stau2-IPs (Figure 4A) conrming
the LC-MS data.
Additionally, the RNA helicase Upf1 (Regulator of nonsense
transcript or Rent), which is not part of the EJC core, but an inte-
gral component of the nonsense-mediated mRNA decay (NMD)
machinery (Le Hir and Se raphin, 2008), was also identied in
both Btz- and Stau2-RNPs. The presence of the EJC and Upf1
indicates that the mRNAs found in Btz-containing RNPs have
not yet undergone translation and are translationally repressed.
The fact that the EJC is not found in the Stau2-RNPs suggests
that the transcripts in these RNPs have already undergone
translation, or alternatively, that another mechanism of transla-
tional control is active in this case. To further validate this nding,
we performed pull-down experiments using brain extracts and
GST-Btz or GST-Stau2 as bait. Whereas eIF4AIII bound to
GST-Btz, it did not bind to GST-Stau2 (Figure 4B) supporting
our previous results. Finally, we performed reverse IPs using
eIF4AIII antibodies and soluble S100 extracts and conrmed
the interaction between eIF4AIII and Btz (Figure 4C). However,
under these conditions, we also detected an interaction between
eIF4AIII and Stau2. Interestingly, when we repeated these
experiments in the presence of the nonhydrolysable ATP analog,
AMP-PNP, to stabilize the binding of the Y14/Magoh hetero-
dimer (Ballut et al., 2005), we could still only detect eIF4AIII,
but not Y14/Magoh in Stau2-RNPs (Figure S3). Although further
work is necessary, our ndings suggest that at least some
portion of Stau2 is bound to eIF4AIII, possibly independently of
the EJC.
Btz- and Stau2-RNPs Contain the Nuclear Cap-Binding
Protein CBP80
A series of studies have reported the presence of general trans-
lation factors in neuronal RNPs (Banerjee et al., 2009; Kanai
et al., 2004; Krichevsky and Kosik, 2001; Tiedge and Brosius,
1996). Therefore, we looked for translational factors in Btz- and
Stau2-RNPs. Three initiation factors in Btz- and two in Stau2-
RNPs (Table 1; Figure 2D) were identied by LC-MS. When we
validated this by IP and western blot, we did not detect any of
the following translation factors in either Btz- or Stau2-RNPs:
eEF1A, eIF1A, eIF4G1, eIF3B1, or eIF4E (Figure 5A). This is in
line with previous ndings for IMP1 (ZBP1) granules (Jnson
et al., 2007). Given the absence of eIF4E and the presence of
Figure 3. Differential Enrichment of Dendritically Localized mRNAs
in Btz- and Stau2-RNPs
Analytical-scale IPs were performed from pooled Optiprep gradient fractions
(F5-8 using Stau2 or F4-7 using Btz antibodies). Control IPs using corre-
sponding PIS were performed in parallel. RNA was isolated, and cDNA was
synthesized. The enrichment of candidate dendritic mRNAs in the Btz- and
Stau2-RNPs was determined by qRT-PCR analysis. Enrichment is calculated
as the levels of a given gene in the immunoprecipitated material relative to
the IP input, and then normalized for background from the control IP.
Error bars represent the SEM from three independent experiments. Students
t test was used to calculate statistical signicance between Stau2 and Btz.
*p < 0.05; **p < 0.01; n.s., statistically not signicant. See also Figure S3 and
Tables S3 and S5.
Cell Reports 5, 17491762, December 26, 2013 2013 The Authors 1753
the EJC, at least in the Btz-RNPs, we then focused on the nuclear
cap-binding protein 80 (CBP80) (di Penta et al., 2009; Hwang
et al., 2010). CBP80 is part of the cap-binding complex (CBC),
which interacts with Upf1 and plays an important role in NMD
and translation. It is thought that the EJC is removed if there
are no premature termination codons. Following this, the CBC
is replaced by eIF4E and translation can proceed (Kim et al.,
2009). Thus, CBP80 and eIF4E are mutually exclusive on a given
mRNA. In line with previous ndings from other labs (di Penta
et al., 2009; Jnson et al., 2007), we found that both (dendritic)
Btz- and Stau2-RNPs contain CBP80 (Figure 5A). Likewise, the
nuclear PABP, PABPN1, is removed prior to cytoplasmic
translation and yet is present in both Stau2- and Btz-RNPs
(Table S1). Together, these ndings provide another line of
evidence that mRNAs present in both neuronal RNPs are trans-
lationally repressed.
To visualize the interaction between CBP80 and Stau2 in
mature neurons, we performed immunouorescence micro-
scopy using antibodies directed against either CBP80 or eIF4E
(labeled in green) and Stau2 (labeled in magenta). Whereas
both CBP80 and eIF4E are expressed in mature hippocampal
neurons, only CBP80but not eIF4Eshows signicant coloc-
alization with Stau2 in dendrites (Figures 5B5D). However,
eIF4E is present at synapses of mature hippocampal neurons
(Figure 5C; (Vessey et al., 2010). This indicates that the predom-
inantly nuclear CBP80 stays associated with Stau2-RNPs
during localization into dendrites of hippocampal neurons.
Furthermore, these experiments show that at least some of the
identied Stau2 interactors are indeed found in dendritic
Stau2-RNPs near synapses.
In addition to general translation factors, ribosomal proteins
and even entire ribosomes have been reported to be present
in neuronal RNA granules (Elvira et al., 2006; Kanai et al.,
2004; Krichevsky and Kosik, 2001). Our systems approach
revealed most of the ribosomal proteins as nonspecic inter-
actors, because they were also found in the control IPs using
PIS (see Table S2, Negatives). However, six ribosomal
proteins were specically associated with Btz- and seven
Btz
eIF4AIII
Magoh
Y14
Upf1
Btz-IP PIS-IP Stau2-IP
Btz-IP Stau2-IP
PIS-IP
RE GE RE GE RE GE RE GE S20 F4-8
EJC
MS
- -
-
-
-
-
4
2
18
17
A
C
B
GST-
Stau2 GST
eIF4AIII
S100 BL u.b.
GST-
Btz
GST-
Stau2 GST
empty
beads
170
130
95
55
26
Pulldown
S100 BL eIF4AIII u.b.
Btz
Stau2
62
59
52
eIF4AIII
empty
beads
IP
GST-
Btz
56
GST-Stau2
GST
GST-Btz
Figure 4. Components of the EJC Are Pre-
sent in Btz-, but Not in Stau2-RNPs
(A) Analytical-scale IPs were performed from
pooled Optiprep gradient fractions (F48) using
Stau2 or Btz antibodies. Control IPs using
corresponding PIS were performed in parallel.
Proteins were eluted rst with RNase A/T1
(RNase Eluate or RE) followed by low pH (glycine
pH 2.5; Glycine Eluate or GE). Both eluates were
separated by SDS-PAGE and analyzed by west-
ern blotting with the antibodies indicated on
the left. The four EJC core components were
detected in the Glycine Eluates of Btz IPs, but not
in the Stau2 IPs. The EJC-associated RNA
helicase Upf1 is present in the RNase Eluate
of both Btz and the Stau2 IPs. The table on the
right summarizes the number of peptide hits
found by MS analysis in Btz and/or Stau2 IPs
(see Table S1 for a complete interactor list).
Peptide hits were added from both RNase and
Glycine Eluates.
(B) GST-pull-down experiments using Btz or
Stau2 as bait. Equal amounts of GST-Btz
FL
, GST-
Stau2
62
and GST protein were separated by
SDS-PAGE and analyzed by western blot for GST
(left panel). Pull-down experiments were per-
formed from cleared soluble E17 rat brain lysate
using GST-Btz and GST-Stau2 chemically
crosslinked to glutathione Sepharose beads.
Control pull-down experiments using GST-gluta-
thione beads as well as empty beads were per-
formed in parallel. Proteins were eluted with low
pH (glycine pH 2.5) and separated by SDS-PAGE.
Western analysis shows that eIF4AIII is present in
eluates of the Btz-, but not of the Stau2-pull-
down.
(C) Analytical scale IPs were performed from
cleared soluble lysate using mouse monoclonal
eIF4AIII antibodies chemically crosslinked to pro-
tein G Sepharose or using empty protein G
Sepharose as control. Proteins were eluted with low pH. Both eluates were separated by SDS-PAGE and analyzed by western blotting with antibodies specic
for the proteins indicated on the left. Both Btz and Stau2 are detected in eluates of the eIF4AIII-IP (see also Figure S3).
BL, brain lysate; u.b., unbound; empty beads, beads without antibodies. See also Table S4.
1754 Cell Reports 5, 17491762, December 26, 2013 2013 The Authors
with Stau2-RNPs (Table S1, Positives; Figure 2D). Interest-
ingly, all ribosomal proteins identied are part of the large
ribosomal subunit. This is of particular note because stress
granules selectively contain components of the small subunit
of the ribosome (Anderson and Kedersha, 2008). To further
investigate this, we performed western blots following IPs using
specic antibodies against the large ribosome subunit protein,
L7a, which has been previously reported to directly interact
with Stau2 (Duchane et al., 2002), as well as antibodies against
the small subunit proteins, S3 and S6. Interestingly, all three
ribosomal proteins were enriched in both neuronal RNPs
(Figure S4). Note, however, that all three ribosomal proteins
mentioned above were also present in the PIS IP (Table S2,
Negatives). Taken together, our data suggest that the
isolated RNPs contain translationally repressed mRNAs as
CBP80 and PABPN1 are all present but eIF4E, and the vast
majority of ribosomal proteins are absent.
A Series of Translational Repressors Associate
with Both RNPs
To further investigate the mechanisms of translational control in
these neuronal particles, we looked for factors with a known
or assumed function in translational regulation. Analysis of the
LC-MS data revealed a noticeable enrichment of a series of
translational regulators (Table S1, Positives; Figures 2D and
6). Some translational regulators preferentially associate with
either Btz- or Stau2-RNPs, whereas others are present in both
types of RNPs. Many of them, e.g., FMRP, Pur-a, and DDX6,
have previously been implicated in RNA localization (Costa-
Mattioli et al., 2009).
We therefore performed analytical IPs to validate the presence
of the following four RBPs: FMRP, Pur-a, DDX6 (Rck, p54), and
RBM14. We also included Pumilio2 (Pum2), because it had been
identied as part of dendritic RNPs near synapses (Vessey et al.,
2006), although it was not detected by LC-MS. All ve RBPs
were present in both Btz- and Stau2-RNPs (Figure 6A). Besides
these, a substantial number of RBPs were found in the negative
interactor list (see Table S2, Negatives) that had been previ-
ously reported as components of neuronal RNA granules: e.g.,
ZBP1 (IgfII-mRBP1/IMP1), hnRNP-Q (Syncrip), hnRNP-U, YB1,
and HuR. They were all enriched in the isolated RNPs, although
there were low levels in the control IP as well.
With the exception of HuR that selectively associates with
Stau2-RNPs, all four other RBPs were found in both Btz- and
Stau2-containing RNPs. Because PABP1 has been reported to
localize in cytoplasmic mRNPs containing untranslated mRNAs,
its presence in both Btz- and Stau2-containing neuronal RNA
granules indicates translational repression. Together, our data
support the hypothesis that RNA transport is coupled to transla-
tional control (Hu ttelmaier et al., 2005).
This prompted us to search for ZBP1 and FMRP, two RBPs
with a known role in translational regulation. As described above
for eIF4AIII, we performed reverse IPs using soluble S100
fractions and ZBP1 and FMRP antibodies and conrmed the
interaction of ZBP1 (Figure 6B) and FMRP (Figure 6C) with Btz
and Stau2.
To validate the association of Stau2 and Btz with Pum2, we
transfected mature hippocampal neurons with EGFP-Pum2
(in green) and then performed immunostaining using antibodies
directed against either Btz or Stau2 (labeled in magenta).
Importantly, the pattern of EGFP-Pum2 closely resembles
immunostaining of endogenous Pum2 in mature hippocampal
neurons (Vessey et al., 2010; Vessey et al., 2006). Interestingly,
approximately 27% versus 39% of EGFP-Pum2 colocalizes
with both Btz- and Stau2-RNPs in distal dendrites, respectively
(Figures 6D6F). This is consistent with recent ndings that
Stau2 and Pum2 form a complex that asymmetrically localizes
target mRNAs in neural precursor cells (Vessey et al., 2012).
Taken together, these data indicate that a large number
of translational repressors are present in both Stau2- and
Btz-RNPs keeping them in a translationally stalled state.
Furthermore, our data clearly show that Pum2 is found in both
dendritic Btz- and Stau2-containing RNPs near synapses
thereby suggesting a possible interaction between these RBPs.
RISC Components Are Found in Both
Btz- and Stau2-RNPs
In recent years, there has been mounting evidence that miRNAs
play critical roles in neurons and at the synapse (Konecna et al.,
2009). Interestingly, the LC-MS data from the Stau2- and
Btz-RNPs revealed the presence of selective RNA-induced
silencing complex (RISC) components in our neuronal RNA
granules: e.g., Mov10, Ago2, and PACT (Table S1; Figures 2D
and 7A). Indeed, colocalization experiments using EGFP-
Mov10 (in green) and antibodies directed against either Btz
or Stau2 (in magenta) conrm that approximately 25% versus
45% of Mov10 is present in both Btz- (Figures 7B and 7D)
and Stau2-RNPs (Figures 7C and 7D) in distal dendrites,
respectively.
In conclusion, our results identied a series of interesting
interaction partners for both Btz and Stau2 with possible roles
in dendritic mRNA localization, translational control and mRNA
decay among others. Furthermore, we present several inde-
pendent lines of evidence that neuronal RNA granules are
much more heterogeneous and possibly dynamic than previ-
ously suggested. Finally, our data suggest that both Btz- and
Stau2-containing RNPs are translationally repressed.
DISCUSSION
Here, we report the interactome of two distinct neuronal RNA
granules isolated from rat brain. The identication of 84 Btz
and 65 Stau2 interactors allowed us to systematically compare
different types of RNPs and draw important conclusions. First,
only one-third of the proteins are shared arguing that neuronal
RNA granules are much more heterogeneous than previously
anticipated. This is not only true on the protein level, but also
on the RNA level (Figure 3; Heraud-Farlow et al., 2013; data
not shown). Our biochemical approach allowed us to identify
highly conserved members of RNPs by integrating previous
proteomes, mostly from cell lines (references in Table S4): e.g.,
FMRP, the RNA helicase Upf1, the cap-binding protein CBP80,
Pur-a, and Mov10. This includes RNA helicases (Jankowsky,
2011) that have previously been implicated with dendritic
mRNA transport (Ashraf et al., 2006; Elvira et al., 2006; Giorgi
et al., 2007; Kanai et al., 2004). Second, neuronal RNA granules
Cell Reports 5, 17491762, December 26, 2013 2013 The Authors 1755
Stau2 eIF4E Merge
Btz-IP PIS-IP Stau2-IP PIS-IP
RE GE RE GE RE GE RE GE S20 F4-8 Btz-IP Stau2-IP
MS
- -
-
-
-
-
-
-
-
- -
-
A
CBP80 Merge
C anti-Stau2 anti-eIF4E (Merge) anti-Stau2 anti-CBP80 (Merge) B
Stau2
eEF1A
eIF1A
eIF4G1
eIF3B1
eIF4E
CBP80
D
s
e
l
c
i
t
r
a
p

2
u
a
t
S

%
eIF4E CBP80
Figure 5. Translational Factors Are Absent, whereas CBP80 Is Present in Both Btz- and Stau2-RNPs
Analytical-scale IPs using Btz or Stau2 antibodies were performed as described in Figure 4.
(A) Known translation factors are absent in both Stau2 and Btz IPs. The nuclear cap-binding protein 80 (CBP80), however, is present in both Stau2 and Btz IPs
suggesting that the RNAs in both Btz- and Stau2-RNPs are translationally stalled. The table on the right summarizes the number of peptide hits by MS analysis
in Btz and/or Stau2 IPs (see Table S1 for a complete list). Note that ribosomal proteins are mostly absent (see Figure S4).
(B and D) CBP80 is present in dendritic Stau2-RNPs. Eight days in vitro (DIV) dissociated hippocampal neurons were immunostained with mouse anti-Stau2
(in magenta) and rabbit anti-CBP80 (in green).
(legend continued on next page)
1756 Cell Reports 5, 17491762, December 26, 2013 2013 The Authors
only share very few, selected components with P-bodies and
stress granules (Anderson and Kedersha, 2006; Zeitelhofer
et al., 2008). This conrms a previous hypothesis postulating
the existence of various distinct types of neuronal RNA granules
(Anderson and Kedersha, 2006). However, it is very likely that
individual components might be shared among them or that
dynamic remodeling of certain neuronal RNA granules might
occur. Third, our data provide experimental support for the
previously formulated hypothesis that mRNA localization and
translational control are tightly linked (Hu ttelmaier et al., 2005).
Interestingly, eIF4AIII, a component of the EJC, regulates
expression of Arc mRNA at the synapse (Giorgi et al., 2007),
supporting our ndings that EJC members associate with Btz-,
but not with endogenous Stau2-RNPs isolated from rat brain.
The presence of the EJC in Btz-RNPsbut not in Stau2-
RNPsalso suggests that the bound targets have not yet under-
gone translation. Because Arc mRNA has one conserved intron
in its 3
0
UTR (Giorgi et al., 2007), it is tempting to speculate that
Btz and the other EJC components assemble at the intron-exon
boundary thereby exerting regulatory functions in addition to
its role in NMD, e.g., regulating protein expression or possibly
dendritic mRNA localization. A recent study proposes that
Btzby interacting with eIF3activates translation in fast
growing cell lines (Chazal et al., 2013). Fourth, the presence of
CBP80 in both (dendritic) Btz- and Stau2-RNPs argues that
the bound RNAs are translationally stalled. Such regulation of
translation has also been found for other types of RNPs (Chiu
et al., 2006; di Penta et al., 2009; Jnson et al., 2007).
Finally, we identify a series of interactors that are known
translational regulators, e.g., Pum2, Mov10, and FMRP among
others. A genetic screen for memory mutants in Drosophila had
identied Stau and Pum (Dubnau et al., 2003). It is therefore
tempting to speculate that both RBPs exert important functions
in dendritic RNPs as previously suggested (Vessey et al., 2010,
2012). Mov10 is an RNA helicase that is part of the RNA-
induced silencing complex (Meister et al., 2005) and that is
involved in repressing CaMKIIa mRNA at the synapse (Banerjee
et al., 2009).
In conclusion, this work identies the molecular machinery
underlying two types of neuronal RNPs. Furthermore, we provide
insights into RNA localization in neurons as our data argue
strongly for neuronal RNA granule heterogeneity. Finally, we
identify a series of translational regulators that are likely to
keep transcripts translationally stalled during transport to their
nal location in neurons.
EXPERIMENTAL PROCEDURES
Reagents Used in This Study
All antibodies, constructs, antigens, and GST-tagged proteins (for pull-down
experiments) used in this study are described in the Supplemental Experi-
mental Procedures.
Preparation of Rat Brain Homogenate
Forty-ve brains from 17-day-old rat embryos (E17) were homogenized on ice
in 5 ml extraction buffer (EB, 25 mMHEPES [pH7.3], 150 mMKCl, 8%glycerol,
0.1% NP-40, 1 mM DTT, protease inhibitor cocktail [Roche], 40 U/ml RNase
inhibitors [Ribolock, Fermentas]), using a motor-driven dounce homogenizer
applying two strokes at 200 rpm and 12 strokes at 1,000 rpm. Brain lysates
were centrifuged at 20,000 3 g for 15 min at 4

C to separate cell debris and

nuclei from the soluble S20 supernatant. Similar results were obtained using
brains from young adult rats (data not shown).
For GST pull-downs or for FMRP-, ZBP1- and eIF4AIII-IPs, brain lysate was
used after high-speed centrifugation. In brief, the soluble lysate was centri-
fuged two times at 20,000 3 g for 5 min at 4

C and one time at 100,000 3 g

for 30 min at 4

Cto yield an S100 brain lysate. For IPs and pull-downs, proteins
were diluted with 1 3 EB to a nal concentration of 14 mg/ml.
Optiprep Density Gradient Centrifugation
Soluble supernatant (1.7 ml) of homogenized brain lysate (S20) was layered on
top of a 10 ml 15%30% linear Optiprep (Axis Shield) density gradient and
centrifuged in a swinging bucket rotor (SW41, Beckman Coulter Genomics)
at 280,000 3 g at 4

C for 2.5 hr. Eleven 900 ml fractions were collected from

the top to the bottom of the gradient, frozen in liquid nitrogen, and stored
at 80

C. Samples of 10 ml of each fraction were boiled in SDS protein gel

sample buffer and subjected to western blotting. To disrupt RNPs, lysates
were treated with 100 mg RNase A/T1 mix for 30 min at room temperature
(RT) prior to Optiprep density gradient centrifugation.
Immunoprecipitations
For preparative IPs, 300 mg of afnity-puried Btz and Stau2 antibodies or
equivalent amounts of corresponding preimmune sera (PIS) were chemically
crosslinked to 300 mg protein A Sepharose (PAS) beads (GE Healthcare/
Amersham). Antibody-bound Sepharose beads were equilibrated with 0.2 M
triethanolamine (pH 9.5) (TEA buffer), and crosslinking was done using
40 mM dimethyl-pimelimidate (DMP) in 200 mM triethanolamine (pH 9.5)
(3 3 45 min at RT). After each crosslinking step, beads were washed for 2 3
10 min in 200 mM ethanolamine (pH 8) at RT. The same amount of PAS beads
without antibodies served as additional negative control (data not shown).
Antibody-bound PAS beads were blocked with 300 mg of yeast tRNAs (Roche)
and 1.5 mg BSA (Fermentas) in 1 3EB for 60 min rotating at 4

C. Pooled RNP-
containing gradient fractions (F4-6 for Btz, F5-7 for Stau2) were precleared
with PAS for 1 hr and allowed to bind to antibody-coupled beads for 90 min
at 4

C. Beads were washed four times with 1 3 EB and two times with PBS
containing 1 mM MgCl
2
. Proteins were eluted with 600 ml of elution buffer con-
taining RNases (200 mg/ml RNase A/T1 [Fermentas] in PBS [pH 7.1]) for 54 min
at RT. After additional washing steps in EB and H
2
O, the remaining bound
proteins were eluted using a low pH buffer (0.2 M glycine pH 2.5) for 10 min
rotating at RT and immediately neutralized in 1 M Tris-HCl (pH 8.5). Proteins
eluted with either RNases or glycine were processed for LC-MS. Samples
were alkylated with iodoacetamide and separated on a 4%12% bis-Tris gel
(NuPAGE, Invitrogen). After visualization of the proteins by silver staining,
entire lanes were sliced into 20 pieces and digested in situ with modied
porcine trypsin (Promega) essentially as described (Shevchenko et al.,
1996). Prior to analysis by nanoLC-MSMS, tryptically digested samples were
puried and concentrated via customized reversed-phase columns adapted
from Rappsilber et al. (2003).
Analytical IPs were performed as described above, except that 100 mg of
antibodies or equivalent amounts of the corresponding PIS were crosslinked
to 50 mg of PAS. S20, S100 brain lysates, or pooled gradient fractions (F4-8)
were used as indicated and 2 mM AMP-PNP was added before the IP where
indicated (Figure S3). Proteins eluted with RNases or glycine were precipitated
(C and D) In contrast, eIF4E that competes with CBP80 for cap binding shows very little colocalization with Stau2 in dendritic RNPs. Seven DIV neurons
were immunostained with rabbit Stau2 (in magenta) and mouse eIF4E antibodies (in green). Boxes represent magnication of depicted areas. Arrowheads
mark colocalization events. Scale bar: 10 mm. (D) Graph showing the percentage of Stau2 particles colocalizing with CBP80 or with eIF4E in dendrites (n = 18,20 or
n = 33,31, respectively). ***Statistically signicant (p < 0.001).
See also Figure S4 and Table S4.
Cell Reports 5, 17491762, December 26, 2013 2013 The Authors 1757
Btz-IP PIS-IP Stau2-IP PIS-IP
RE GE RE GE RE GE RE GE S20 F4-8
A
Btz-IP Stau2-IP
MS
FMRP 2 3 7.3 2.7 28.2 24.9
Pum2 - - 6.1 10,6 7.4 8.7
2 3 Pur- 1.4 2.0 36.3 13.9
DDX6 3 - 5.3 4.6 9.1 0.1
RBM14 - 19 5.0 0.4 4.2 4.2
*HuR 3 - n.d. n.d. 4.4 6.3
*hnRNPQ 6 8 9.1 9.9 4.0 5.6
7 11 *ZBP1 1.0 1.4 3.8 3.4
*hnRNPU 7 3 14.8 11.9 8.2 15.3
*YB1 - 11 12.9 0.4 1.7 2.4
PABP1 18 32 5.5 1.6 6.9 9.5
U5-116kD 5 - 5.7 2.4 17.2 11.6
Btz Stau2
PIS / IP PIS / IP
B
C
IP
S100 BL ZBP1 u.b.
Btz
Stau2
62
59
52
ZBP1
empty
beads
anti-Btz EGFP-Pum2 (Merge)
Merge
D
EGFP-
Pum2
Btz
anti-Stau2 EGFP-Pum2 (Merge)
E
Stau2 Merge EGFP-
Pum2
IP
S100 BL FMRP u.b.
empty
beads
Btz
Stau2
62
59
52
FMRP
F
Btz Stau2
s
e
l
c
i
t
r
a
p
2
m
u
P
-
P
F
G
E
%
Figure 6. A Series of Translational Repressors Enrich in Both Btz- and Stau2-RNPs
(A) Analytical-scale IPs using Btz or Stau2 antibodies were performed as described in Figure 4. (A) shows the presence of translational repressors FMRP, Pum2,
Pur-a, DDX6, RBM14, ZBP1, hnRNP Q(Syncrip), hnRNP U, and YB1 in both Btz and Stau2 precipitates, whereas HuRis only in the Stau2 eluate. The presence of
PABP1 in both eluates indicates the presence of mRNA in both Btz- and Stau2-RNPs and the intactness of the RNA granules. In the box in the middle of the panel,
protein levels in the Btz and Stau2 IPs were quantied in comparison to their corresponding PIS IP. Band intensities were measured with ImageJ. The values
represent the percentage of the PIS to the signal and are the mean SD of three to ve experiments (PIS/IP, see Experimental Procedures for details). With the
(legend continued on next page)
1758 Cell Reports 5, 17491762, December 26, 2013 2013 The Authors
as described (Wessel and Flu gge, 1984) and analyzed by western blotting.
Protein levels were quantied using ImageJ. Values shown in Figure 6A repre-
sent the percentage of background (PIS) to signal (IP) and are the mean SD
of three to ve experiments. Please note that, in some cases, background
levels could not be determined, therefore preventing us from expressing
values as IP/PIS.
For IP of RNAs and qRT-PCR, 100 mg of afnity-puried Btz or Stau2 an-
tibodies and equal amounts of the corresponding PIS were individually
coupled to 50 ml of PAS beads (Sigma) and blocked as above. Pooled
RNP-containing gradient fractions were precleared with PAS, and 50 ml of
the precleared gradient fractions was removed for RNA extraction to serve
as input control. The remaining precleared fractions were divided equally
exception of U5-116 kDa and ZBP1 (both were not detectable in the control lanes), levels of the RNase Eluate were quantied. The table on the right summarizes
the number of peptides found by MS analysis in Btz and/or the Stau2 IPs (see Table S1 for a complete list). Peptide hits were added together from both RNase
and Glycine Eluates. Proteins labeled by an asterisk were originally identied in the corresponding PIS-IPs (see negative interactor list, Table S2).
(B and C) The translational regulatory factors, ZBP1 and FMRP, are enriched in both Btz- and Stau2-RNPs. Analytical IPs using mouse monoclonal ZBP1 (B) or
mouse monoclonal FMRP (C) antibodies were performed as in Figure 4 and analyzed by western blotting. Both Btz and Stau2 are detected in the eluates of both
the ZBP1- and the FMRP-IP.
(DF) Pum2 shows signicant colocalization with both Btz and Stau2 in dendritic RNPs. Fourteen DIV (D) and 8 DIV (E) dissociated hippocampal neurons were
transfected with EGFP-Pum2 (in green) and immunostained with either rabbit Btz (D) or rabbit Stau2 (E) (both in magenta) antibodies. Boxes represent
magnication of depicted areas; arrowheads mark colocalization events. Scale bar: 10 mm. (F) Graph showing the percentage of EGFP-Pum2 particles
colocalizing with Btz or with Stau2 in dendrites (n = 51 or n = 47, respectively). ***Statistically signicant (p < 0.001).
See also Table S4.
C B
anti-Btz EGFP-Mov10 (Merge)
A
Btz-IP PIS-IP Stau2-IP PIS-IP
RE GE RE GE RE GE RE GE S20 F4-8 Btz-IP Stau2-IP
MS
Mov10
- 4
Ago2 2 -
*PACT 6 6
Btz Merge EGFP-
Mov10
anti-Stau2 EGFP-Mov10 (Merge)
Stau2 Merge EGFP-
Mov10
D
Btz Stau2
s
e
l
c
i
t
r
a
p

0
1
v
o
M
-
P
F
G
E

%
Figure 7. Selected RISCProteins Are Found
in Both Dendritic Btz- and Stau2-RNPs
(A) Components of the RNA-induced silencing
complex (RISC), Mov10, Ago2, and PACT are
present in both Btz- and Stau2-RNPs. Analytical
IPs using Btz or Stau2 antibodies were performed
as described in Figure 4. PACT was originally
identied in the corresponding PIS-IPs (see
negative interactor list, Table S2).
(BD) Mov10 shows signicant colocalization with
both Btz (B) and Stau2 (C) in dendritic RNPs. Nine
DIV (B) or 11 DIV (C) dissociated hippocampal
neurons were transfected with EGFP-Mov10 (in
green) and immunostained with either rabbit Btz
(B) or rabbit Stau2 (C) (both in magenta) anti-
bodies. Boxes represent magnication of depicted
areas. Arrowheads mark colocalization events.
Scale bar: 10 mm. (D) Graph showing the per-
centage of EGFP-Mov10 particles colocalizing
with Btz or Stau2 in dendrites (n = 46 or n = 54,
respectively). ***Statistically signicant (p < 0.001).
See also Table S4.
among the coupled beads and allowed to bind
for 90 min rotating at 4

C. Beads were washed

2 3 EB (with 0.5% NP40), 2 3 EB (with 0.1%
NP40), and 2 3 5 mM Tris (pH 7.4), 100 mM
NaCl. Total RNA was isolated directly from the
beads and from the input control using a
mirVana miRNA Isolation Kit (Ambion). 0.5 mg of
input RNA, 0.5 mg of IP RNA, and an equal vol-
ume of PIS RNA was taken as templates for
random hexamer primed cDNA synthesis using
SuperScript III reverse transcriptase (Invitrogen)
in a 20 ml reaction volume. cDNAs were diluted
1:10, and 3 ml was used to perform qRT-PCR
in a 25 ml nal volume using IQ SYBR Green
Supermix (Bio-Rad) and gene-specic primers
(Table S5) on a MyiQ iCycler (Bio-Rad) according
to the manufacturers instructions. Quantication of gene expression was
calculated according to the DDCt method using the average of two inde-
pendent reference genes. Experiments were repeated with RNA isolated
from three independent IPs. Statistical signicance using a Students t
test was considered signicant if p < 0.05.
Liquid Chromatography Mass Spectrometry and Data analysis
Three independent IP experiments for Stau2 were analyzed and two inde-
pendent IP experiments for Btz. Samples were analyzed by data-dependent
nanocapillary reversed-phase LC-MSMS using customized 75 mminner diam-
eter columns packed with C18 3 mm diameter Reprosil beads (Maisch) on a
nanoLC system (Agilent Technologies) coupled to a quadrupole time-of-ight
Cell Reports 5, 17491762, December 26, 2013 2013 The Authors 1759
(QTOF) mass spectrometer (QTOF Premier or QTOF Ultima, Waters). Data-
dependent acquisition was performed for 60 min using one MS channel for
every three MSMS channels and a dynamic exclusion for selected ions of 60 s.
Proteins were identied by automated database searching (Mascot, Matrix
Science) against the rat and mouse International Protein Index protein
sequence databases (IPI, versions 3.21 and 3.26, European Bioinformatics
Institute, http://www.ebi.ac.uk/IPI). This compilation of entries from Swiss-
Prot, TrEMBL, RefSeq, and Ensembl was appended with human keratin pro-
teins. Search parameters were as follows: MS and MSMS tolerances of
20 or 15 ppm and 0.1 or 0.05 Da, respectively, tryptic specicity allowing
for 1 missed cleavage site, xed modication of carbamidomethylation of
cysteine residues, and variable modication of oxidation of methionine
residues. Criterion for a positive protein identication was identication of a
minimum of two peptides of length of six or more amino acids with a Mascot
peptide score of R20. A false-positive detection rate of <1% was estimated
by searching a reversed database. Protein grouping on the basis of shared
peptides was realized by an in-house Perl script.
Gene ontology (GO) termanalysis was performed using DAVID(http://david.
abcc.ncifcrf.gov). To be able to search for rat and mouse proteins at the same
time, the ofcial gene symbols (gene name) were selected for the identied
proteins from the UniProtKB/Swiss-Prot database (http://www.ebi.ac.uk/
swissprot). In DAVID, the following functional categories were analyzed for
both Btz and Stau2: biological pathway (BP), cellular compartment (CC),
molecular function (MF), as well as interpro and kegg to identify protein do-
mains. In Figure 2D, proteins with known functions that have been overlooked
by GO term were added or false annotations deleted (corrected).
GST-Pull-Downs
Thirty micrograms of GST-Btz
FL
, GST-Stau2
FL
or GST proteins was bound and
crosslinked to 30 ml of glutathione Sepharose following the same protocol as
for crosslinking antibodies to PAS. Brain lysate was centrifuged at high speed
as described above and diluted in 1 3 EB to obtain a protein concentration
of approximately 14 mg/ml. Brain lysates were precleared with glutathione
Sepharose beads by rotating them 1 hr at 4

C. Precleared brain lysate was

incubated with the beads for 90 min rotating at 4

C. Approximately 1 ml of
brain lysate was used for each pull-down experiment. Beads were washed
four times with 1 3 EB and two times with ddH
2
O, and proteins were eluted
in 120 ml low pH buffer (0.2 M glycine pH 2.5) for 10 min rotating at RT and
neutralized in 30 ml 1 MTris-Cl (pH8.5). Eluted proteins were precipitated using
chloroform/methanol as described above and analyzed by western blotting.
Hippocampal Cultures, Transient Transfections, and
Immunostainings
Rat hippocampal neurons were cultured, transiently transfected, and immuno-
stained as described (Goetze et al., 2006). Figure legends indicate the
respective age of neurons in culture. For quantifying colocalizing events, a
set of imageseither double immunouorescence stainings or immunostain-
ings of hippocampal neurons that were transfected with GFP-tagged Pum2
or Mov10were selected and manually scored by at least two independent
observers in a blind manner. Colocalizing events of either Btz or Stau2 with
EGFP-Pum2 or EGFP-Mov10 were conrmed by linescan (MetaMorph 6.3,
Universal Imaging). Statistical analysis was performed using Prism(GraphPad)
and considered signicant if p < 0.05.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures,
four gures, and ve tables and can be found with this article online at
http://dx.doi.org/10.1016/j.celrep.2013.11.023.
ACKNOWLEDGMENTS
This manuscript is dedicated to Julia Sandholzer for her excellent scientic
contributions. We thank Franziska Busch, Inge Kepert, Ingrid Kieweg,
Bastian Popper, and Sabine Spath for assistance, Drs. Egon Ogris and
Stefan Schuechner for raising mouse monoclonal Stau2 antibodies, and
Drs. Paul Anderson, Dorothee Dormann, Stefan Hu ttelmaier, Eckhard Jankow-
sky, Gunter Meister, and Jernej Ule for discussions. This work was supported
by the Schram-Foundation (T287/14958/2005), the ESF/Eurocores program
RNA Quality (FWF I127-B12), an FWF grant (FWF P20583-B12), the SFB
RNA-Seq (FWF F4314-B09), the Doktoratskolleg RNA Biology (FWF W1207-
B09), a project grant from the HFSP (RGP24/2008), the Herzfelder foundation
(Vienna, Austria), and the Max-Planck Society (all to M.A.K.), a FWF grant
(P19514-B09 to P.M.), and a DOCforte PhD fellowship from the Austrian
Academy of Sciences to Renate Fritzsche.
Received: June 7, 2013
Revised: October 7, 2013
Accepted: November 12, 2013
Published: December 19, 2013
REFERENCES
Amrute-Nayak, M., and Bullock, S.L. (2012). Single-molecule assays reveal
that RNA localization signals regulate dynein-dynactin copy number on
individual transcript cargoes. Nat. Cell Biol. 14, 416423.
Anderson, P., and Kedersha, N. (2006). RNA granules. J. Cell Biol. 172,
803808.
Anderson, P., and Kedersha, N. (2008). Stress granules: the Tao of RNA triage.
Trends Biochem. Sci. 33, 141150.
Andersen, C.B., Ballut, L., Johansen, J.S., Chamieh, H., Nielsen, K.H., Oliveira,
C.L., Pedersen, J.S., Se raphin, B., Le Hir, H., and Andersen, G.R. (2006).
Structure of the exon junction core complex with a trapped DEAD-box
ATPase bound to RNA. Science 313, 19681972.
Ashraf, S.I., McLoon, A.L., Sclarsic, S.M., and Kunes, S. (2006). Synaptic
protein synthesis associated with memory is regulated by the RISC pathway
in Drosophila. Cell 124, 191205.
Ballut, L., Marchadier, B., Baguet, A., Tomasetto, C., Se raphin, B., and Le Hir,
H. (2005). The exon junction core complex is locked onto RNA by inhibition
of eIF4AIII ATPase activity. Nat. Struct. Mol. Biol. 12, 861869.
Banerjee, S., Neveu, P., and Kosik, K.S. (2009). A coordinated local transla-
tional control point at the synapse involving relief from silencing and MOV10
degradation. Neuron 64, 871884.
Batish, M., van den Bogaard, P., Kramer, F.R., and Tyagi, S. (2012). Neuronal
mRNAs travel singly into dendrites. Proc. Natl. Acad. Sci. USA 109, 4645
4650.
Bono, F., Ebert, J., Lorentzen, E., and Conti, E. (2006). The crystal structure of
the exon junction complex reveals howit maintains a stable grip on mRNA. Cell
126, 713725.
Brendel, C., Rehbein, M., Kreienkamp, H.J., Buck, F., Richter, D., and Kindler,
S. (2004). Characterization of Staufen 1 ribonucleoprotein complexes. Bio-
chem. J. 384, 239246.
Chazal, P.E., Daguenet, E., Wendling, C., Ulryck, N., Tomasetto, C., Sargueil,
B., and Le Hir, H. (2013). EJC core component MLN51 interacts with eIF3 and
activates translation. Proc. Natl. Acad. Sci. USA 110, 59035908.
Chiu, Y.L., Witkowska, H.E., Hall, S.C., Santiago, M., Soros, V.B., Esnault, C.,
Heidmann, T., and Greene, W.C. (2006). High-molecular-mass APOBEC3G
complexes restrict Alu retrotransposition. Proc. Natl. Acad. Sci. USA 103,
1558815593.
Costa-Mattioli, M., Sossin, W.S., Klann, E., and Sonenberg, N. (2009).
Translational control of long-lasting synaptic plasticity and memory. Neuron
61, 1026.
di Penta, A., Mercaldo, V., Florenzano, F., Munck, S., Ciotti, M.T., Zalfa, F.,
Mercanti, D., Molinari, M., Bagni, C., and Achsel, T. (2009). Dendritic LSm1/
CBP80-mRNPs mark the early steps of transport commitment and transla-
tional control. J. Cell Biol. 184, 423435.
Doyle, M., and Kiebler, M.A. (2011). Mechanisms of dendritic mRNA transport
and its role in synaptic tagging. EMBO J. 30, 35403552.
1760 Cell Reports 5, 17491762, December 26, 2013 2013 The Authors
Dubnau, J., Chiang, A.S., Grady, L., Barditch, J., Gossweiler, S., McNeil, J.,
Smith, P., Buldoc, F., Scott, R., Certa, U., et al. (2003). The staufen/pumilio
pathway is involved in Drosophila long-term memory. Curr. Biol. 13, 286296.
Duchane, T.F., Hemraj, I., Furic, L., Deitinghoff, A., Kiebler, M.A., and
DesGroseillers, L. (2002). Staufen2 isoforms localize to the somatodendritic
domain of neurons and interact with different organelles. J. Cell Sci. 115,
32853295.
Elvira, G., Wasiak, S., Blandford, V., Tong, X.K., Serrano, A., Fan, X., del Rayo
Sa nchez-Carbente, M., Servant, F., Bell, A.W., Boismenu, D., et al. (2006).
Characterization of an RNA granule from developing brain. Mol. Cell. Prote-
omics 5, 635651.
Giorgi, C., Yeo, G.W., Stone, M.E., Katz, D.B., Burge, C., Turrigiano, G., and
Moore, M.J. (2007). The EJC factor eIF4AIII modulates synaptic strength and
neuronal protein expression. Cell 130, 179191.
Goetze, B., Tuebing, F., Xie, Y., Dorostkar, M.M., Thomas, S., Pehl, U., Boehm,
S., Macchi, P., and Kiebler, M.A. (2006). The brain-specic double-stranded
RNA-binding protein Staufen2 is required for dendritic spine morphogenesis.
J. Cell Biol. 172, 221231.
Harlow, E., and Lane, D. (1988). Antibodies: A Laboratory Manual (Cold Spring
Harbor: Cold Spring Harbor Laboratory Press).
Heraud-Farlow, J.E., Sharangdhar, T., Li, X., Pfeifer, P., Tauber, S., Orozco, D.,
Ho rmann, A., Thomas, S., Bakosova, A., Farlow, A.R., et al. (2013). Staufen2
regulates neuronal target mRNAs. Cell Rep. 5, this issue, 15111518.
Holt, C.E., and Bullock, S.L. (2009). Subcellular mRNA localization in animal
cells and why it matters. Science 326, 12121216.
Hu ttelmaier, S., Zenklusen, D., Lederer, M., Dictenberg, J., Lorenz, M., Meng,
X., Bassell, G.J., Condeelis, J., and Singer, R.H. (2005). Spatial regulation of
beta-actin translation by Src-dependent phosphorylation of ZBP1. Nature
438, 512515.
Hwang, J., Sato, H., Tang, Y., Matsuda, D., and Maquat, L.E. (2010). UPF1
association with the cap-binding protein, CBP80, promotes nonsense-medi-
ated mRNA decay at two distinct steps. Mol. Cell 39, 396409.
Jankowsky, E. (2011). RNA helicases at work: binding and rearranging. Trends
Biochem. Sci. 36, 1929.
Jnson, L., Vikesaa, J., Krogh, A., Nielsen, L.K., Hansen, Tv., Borup, R.,
Johnsen, A.H., Christiansen, J., and Nielsen, F.C. (2007). Molecular composi-
tion of IMP1 ribonucleoprotein granules. Mol. Cell. Proteomics 6, 798811.
Kanai, Y., Dohmae, N., and Hirokawa, N. (2004). Kinesin transports RNA: isola-
tion and characterization of an RNA-transporting granule. Neuron 43, 513525.
Kiebler, M.A., and Bassell, G.J. (2006). Neuronal RNA granules: movers and
makers. Neuron 51, 685690.
Kiebler, M.A., Lo pez-Garca, J.C., and Leopold, P.L. (1999b). Purication and
characterization of rat hippocampal CA3-dendritic spines associated with
mossy ber terminals. FEBS Lett. 445, 8086.
Kim, K.M., Cho, H., Choi, K., Kim, J., Kim, B.W., Ko, Y.G., Jang, S.K., and Kim,
Y.K. (2009). A new MIF4G domain-containing protein, CTIF, directs nuclear
cap-binding protein CBP80/20-dependent translation. Genes Dev. 23, 2033
2045.
Ko hrmann, M., Luo, M., Kaether, C., DesGroseillers, L., Dotti, C.G., and
Kiebler, M.A. (1999). Microtubule-dependent recruitment of Staufen-green
uorescent protein into large RNA-containing granules and subsequent den-
dritic transport in living hippocampal neurons. Mol. Biol. Cell 10, 29452953.
Konecna, A., Heraud, J.E., Schoderboeck, L., Raposo, A.A., and Kiebler, M.A.
(2009). What are the roles of microRNAs at the mammalian synapse? Neurosci.
Lett. 466, 6368.
Krichevsky, A.M., and Kosik, K.S. (2001). Neuronal RNA granules: a link
between RNA localization and stimulation-dependent translation. Neuron 32,
683696.
Le Hir, H., and Se raphin, B. (2008). EJCs at the heart of translational control.
Cell 133, 213216.
Macchi, P., Kroening, S., Palacios, I.M., Baldassa, S., Grunewald, B., Ambro-
sino, C., Goetze, B., Lupas, A., St Johnston, D., and Kiebler, M. (2003).
Barentsz, a new component of the Staufen-containing ribonucleoprotein
particles in mammalian cells, interacts with Staufen in an RNA-dependent
manner. J. Neurosci. 23, 57785788.
Maher-Laporte, M., Berthiaume, F., Moreau, M., Julien, L.A., Lapointe, G.,
Mourez, M., and DesGroseillers, L. (2010). Molecular composition of stau-
fen2-containing ribonucleoproteins in embryonic rat brain. PLoS ONE 5,
e11350.
Mallardo, M., Deitinghoff, A., Mu ller, J., Goetze, B., Macchi, P., Peters, C., and
Kiebler, M.A. (2003). Isolation and characterization of Staufen-containing
ribonucleoprotein particles from rat brain. Proc. Natl. Acad. Sci. USA 100,
21002105.
Martin, K.C., and Ephrussi, A. (2009). mRNA localization: gene expression in
the spatial dimension. Cell 136, 719730.
Martin, S.G., Leclerc, V., Smith-Litie` re, K., and St Johnston, D. (2003). The
identication of novel genes required for Drosophila anteroposterior axis
formation in a germline clone screen using GFP-Staufen. Development 130,
42014215.
Meister, G., Landthaler, M., Peters, L., Chen, P.Y., Urlaub, H., Lu hrmann, R.,
and Tuschl, T. (2005). Identication of novel argonaute-associated proteins.
Curr. Biol. 15, 21492155.
Mikl, M., Vendra, G., and Kiebler, M.A. (2011). Independent localization of
MAP2, CaMKIIa and b-actin RNAs in low copy numbers. EMBO Rep. 12,
10771084.
Palacios, I.M., Gateld, D., St Johnston, D., and Izaurralde, E. (2004).
An eIF4AIII-containing complex required for mRNA localization and
nonsense-mediated mRNA decay. Nature 427, 753757.
Rappsilber, J., Ishihama, Y., and Mann, M. (2003). Stop and go extraction tips
for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS
sample pretreatment in proteomics. Anal. Chem. 75, 663670.
Shevchenko, A., Wilm, M., Vorm, O., and Mann, M. (1996). Mass spectrometric
sequencing of proteins silver-stained polyacrylamide gels. Anal. Chem. 68,
850858.
Sisson, T.H., and Castor, C.W. (1990). An improved method for immobilizing
IgG antibodies on protein A-agarose. J. Immunol. Methods 127, 215220.
St Johnston, D. (2005). Moving messages: the intracellular localization of
mRNAs. Nat. Rev. Mol. Cell Biol. 6, 363375.
Sutton, M.A., and Schuman, E.M. (2006). Dendritic protein synthesis, synaptic
plasticity, and memory. Cell 127, 4958.
Tang, S.J., Meulemans, D., Vazquez, L., Colaco, N., and Schuman, E. (2001). A
role for a rat homolog of staufen in the transport of RNA to neuronal dendrites.
Neuron 32, 463475.
Tiedge, H., and Brosius, J. (1996). Translational machinery in dendrites of
hippocampal neurons in culture. J. Neurosci. 16, 71717181.
Tu bing, F., Vendra, G., Mikl, M., Macchi, P., Thomas, S., and Kiebler, M.A.
(2010). Dendritically localized transcripts are sorted into distinct ribonucleo-
protein particles that display fast directional motility along dendrites of
hippocampal neurons. J. Neurosci. 30, 41604170.
van Eeden, F.J., Palacios, I.M., Petronczki, M., Weston, M.J., and St Johnston,
D. (2001). Barentsz is essential for the posterior localization of oskar mRNA
and colocalizes with it to the posterior pole. J. Cell Biol. 154, 511523.
Vessey, J.P., Vaccani, A., Xie, Y., Dahm, R., Karra, D., Kiebler, M.A., and
Macchi, P. (2006). Dendritic localization of the translational repressor Pumilio
2 and its contribution to dendritic stress granules. J. Neurosci. 26, 64966508.
Vessey, J.P., Schoderboeck, L., Gingl, E., Luzi, E., Rieer, J., Di Leva, F., Karra,
D., Thomas, S., Kiebler, M.A., and Macchi, P. (2010). Mammalian Pumilio
2 regulates dendrite morphogenesis and synaptic function. Proc. Natl. Acad.
Sci. USA 107, 32223227.
Vessey, J.P., Amadei, G., Burns, S.E., Kiebler, M.A., Kaplan, D.R., and Miller,
F.D. (2012). An asymmetrically localized Staufen2-dependent RNA complex
regulates maintenance of mammalian neural stem cells. Cell Stem Cell 11,
517528.
Cell Reports 5, 17491762, December 26, 2013 2013 The Authors 1761
Villace , P., Mario n, R.M., and Ort n, J. (2004). The composition of Staufen-
containing RNA granules from human cells indicates their role in the regulated
transport and translation of messenger RNAs. Nucleic Acids Res. 32, 2411
2420.
Wessel, D., and Flu gge, U.I. (1984). A method for the quantitative recovery
of protein in dilute solution in the presence of detergents and lipids. Anal.
Biochem. 138, 141143.
Zeitelhofer, M., Karra, D., Macchi, P., Tolino, M., Thomas, S., Schwarz, M.,
Kiebler, M., and Dahm, R. (2008). Dynamic interaction between P-bodies
and transport ribonucleoprotein particles in dendrites of mature hippocampal
neurons. J. Neurosci. 28, 75557562.
Zimyanin, V.L., Belaya, K., Pecreaux, J., Gilchrist, M.J., Clark, A., Davis, I., and
St Johnston, D. (2008). In vivo imaging of oskar mRNA transport reveals the
mechanism of posterior localization. Cell 134, 843853.
1762 Cell Reports 5, 17491762, December 26, 2013 2013 The Authors